Detection of FOXL2 C134W Mutation Status by a Novel BaseScope In Situ Hybridization Assay is Highly Sensitive and Specific for Adult Granulosa Cell Tumors.

Adult granulosa cell tumors (AGCTs) are a molecularly distinct group of malignant ovarian sex cord-stromal tumors (SCSTs) characterized by a nearly ubiquitous c.402C>G/p.C134W mutation in FOXL2 (hereafter referred to as "C134W"). In some cases, AGCT exhibits marked morphologic overlap with other SCSTs and has an identical immunophenotype, and molecular testing may be necessary to help confirm the diagnosis. However, molecular testing is time consuming, relatively expensive, and unavailable in many pathology laboratories. We describe the development and validation of an in situ hybridization (ISH) custom BaseScope assay for the detection of the FOXL2 C134W mutation. We evaluated 106 ovarian SCSTs, including 78 AGCTs, 9 juvenile granulosa cell tumors, 18 fibromas (cellular and conventional), and 1 SCST, not otherwise specified, as well as 53 epithelial ovarian tumors (42 endometrioid carcinomas and 11 carcinosarcomas) and 1 STK11 adnexal tumor for the presence or absence of FOXL2 wild-type and FOXL2 C134W RNA expression via BaseScope-ISH. Fifty-one tumors had previously undergone DNA sequencing of the FOXL2 gene. Across the entire cohort, the FOXL2 C134W probe staining was positive in 77 of 78 (98.7%) AGCTs. Two of 81 (2.5%) non-AGCTs also showed positive staining, both of which were epithelial ovarian tumors. The assay worked in tissue from blocks >20 years old. There was 100% concordance between the FOXL2 sequencing and BaseScope-ISH results. Overall, assessment of FOXL2 mutation status by custom BaseScope-ISH demonstrated 98.7% sensitivity and 97.5% specificity for the diagnosis of AGCT. BaseScope-ISH for FOXL2 C134W represents a reasonable alternative to sequencing, is quicker and less expensive, and is more easily incorporated than molecular testing into many pathology laboratories. It also has the advantage of requiring less tissue, and the neoplastic cells can be directly visualized on stained sections.

Uroplakin II (UPII), GATA3, and p40 are Highly Sensitive Markers for the Differential Diagnosis of Invasive Urothelial Carcinoma.

Distinguishing between invasive urothelial carcinoma from other genitourinary lesions such as prostatic and renal carcinomas can be difficult, and may require highly sensitive immunohistochemical markers. GATA-binding protein 3 (GATA3) has been reported in a high percentage of urothelial and breast carcinomas. Mouse monoclonal uroplakin II (UPII) and p40 antibodies have recently been developed and demonstrated high specificity in urothelial carcinoma. This study evaluated the immunohistochemical staining sensitivities of UPII, GATA3, p40, and p63 in the detection of invasive urothelial carcinoma. UPII, GATA3, and p40 were further tested for specificity in lung, breast, colon, kidney, and prostate cancers. In all invasive urothelial carcinoma cases, UPII, GATA3, p40, and p63 exhibited sensitivities of 77.7%, 83.5%, 85.4%, and 80.6%, respectively. The combination of UPII, GATA3, and p40 antibodies stained 94.2% (97/103) of all invasive urothelial carcinoma cases, including 92.2% (71/77) of grade 2-3 urothelial carcinomas. In addition, GATA3 and UPII showed negative staining in lung squamous cell carcinomas and p40 showed negative staining in breast infiltrating ductal carcinomas. The combination of UPII, GATA3, and p40 showed negative staining in lung adenocarcinoma, colon adenocarcinoma, and renal carcinomas. In conclusion, UPII, GATA3, and p40, when used in combination, are highly sensitive in the differential diagnosis of invasive urothelial carcinoma.

A newly developed mouse monoclonal SOX10 antibody is a highly sensitive and specific marker for malignant melanoma, including spindle cell and desmoplastic melanomas.

CONTEXT: Recent immunohistochemical studies have demonstrated Sry-related HMG-Box gene 10 (SOX10) expression in malignant melanomas, malignant peripheral nerve sheath tumors, a subset of breast carcinomas, and gliomas. SOX10 has shown important clinical utility in its ability to detect desmoplastic and spindle cell melanomas. To date, most publications have employed a research use-only goat polyclonal SOX10 antibody for immunohistochemical staining. OBJECTIVE: To describe the development of a new mouse monoclonal SOX10 antibody (BC34) and evaluate its immunohistochemical staining profile in a wide range of normal and neoplastic tissues, with an emphasis on melanoma. DESIGN: SOX10 antibody was optimized for staining using a polymer detection system and visualization with diaminobenzidine. RESULTS: In normal tissues, SOX10 was expressed in skin melanocytes and eccrine cells, breast myoepithelial and lobular epithelial cells, salivary gland myoepithelial cells, peripheral nerve Schwann cells, and central nervous system glial cells. SOX10 was expressed in 238 of 257 melanomas (92.6%), including 50 of 51 of both spindle cell and desmoplastic melanomas (98%). SOX10 was expressed in 100% of nevi (20 of 20) and schwannomas (28 of 28). In other neoplasms, SOX10 was expressed in 18 of 109 invasive ductal breast carcinomas (16.5%). All other carcinomas were negative for SOX10. SOX10 was identified in 25 of 52 central nervous system neoplasms, primarily in astrocytomas (22 of 41; 53.7%), and in 4 of 99 various sarcomas examined (4.0%). CONCLUSIONS: The newly developed mouse monoclonal SOX10 antibody BC34 is highly sensitive and specific for malignant melanoma, including desmoplastic and spindle cell variants, and appears highly suitable for clinical use.

A newly developed uroplakin II antibody with increased sensitivity in urothelial carcinoma of the bladder.

CONTEXT: Uroplakin II is a 15-kDa protein component of the urothelial plaques that enhance the permeability barrier and strength of the urothelium. Studies have shown uroplakin II messenger RNA to be expressed in bladder cancer tissues and peripheral blood of patients with urothelial carcinoma. Little is known about the protein expression of uroplakin II in urothelial carcinoma, possibly because of the absence of a commercially available uroplakin II antibody. Pathologists have used the uroplakin III antibody (AU1) to identify tumors of urothelial origin; however, the use of AU1 is limited because of its poor sensitivity. OBJECTIVES: To evaluate a newly developed mouse monoclonal uroplakin II antibody (BC21) in urothelial carcinoma and to compare it with previously developed mouse monoclonal uroplakin III (BC17 and AU1). DESIGN: Uroplakin II and III antibodies were optimized for staining using a horseradish peroxidase-polymer detection system and were visualized with 3,3'-diaminobenzidine. RESULTS: BC21, BC17, and AU1 demonstrated sensitivities in urothelial carcinoma of the bladder of 79% (44 of 56), 55% (31 of 56) (P = .002), and 34% (19 of 56) (P < .001), respectively. Subsequently, the increased staining sensitivity and intensity of BC21, compared with BC17, was validated in a larger study (134 of 174; 77% and 94 of 174; 54%, respectively) (P < .001). BC21 was found to be highly specific when evaluated in various normal and neoplastic tissues, including prostatic and renal carcinomas. CONCLUSIONS: The mouse monoclonal uroplakin II antibody (BC21) demonstrated superior sensitivity and specificity in urothelial carcinoma, compared with uroplakin III (BC17 and AU1), suggesting its advantages in the differential diagnosis of urothelial carcinoma and in the detection of tumors of unknown origin.

An immunohistochemical analysis of a newly developed, mouse monoclonal p40 (BC28) antibody in lung, bladder, skin, breast, prostate, and head and neck cancers.

CONTEXT: Immunohistochemistry is important to the pathologist for accurate diagnosis of lung cancer. In recent studies, a rabbit polyclonal p40 (RPp40) antibody demonstrated equivalent staining versus anti-p63 in lung squamous cell carcinoma, and superior specificity because it stains a lesser percentage of lung adenocarcinoma. OBJECTIVES: To develop an anti-p40 mouse monoclonal antibody (MMp40) for immunohistochemistry, and to evaluate its sensitivity and specificity in normal and neoplastic tissues, with emphasis on lung cancer. DESIGN: The MMp40 (BC28) antibody was developed and tested for specificity and sensitivity on normal (n = 34) and neoplastic tissues (n = 493). Staining of MMp40, p63, and RPp40 were directly compared in lung cancers, and MMp40 was evaluated in breast, bladder, skin, prostate, and head and neck cancers. Benign prostate glands and prostatic intraepithelial neoplasia were also tested in a direct comparison of MMp40 versus p63. RESULTS: The MMp40 provided equivalent staining versus RPp40 and p63 in lung squamous cell carcinoma, but stained a lesser percentage of lung adenocarcinoma than p63 did. The MMp40 staining was observed in a greater percentage of urothelial carcinomas, squamous and basal cell skin cancers, and head and neck cancers of squamous cell origin. No breast-infiltrating ductal carcinomas or prostatic adenocarcinomas were stained. The MMp40 expression in basal cells of prostate glands and prostatic intraepithelial neoplasia were almost identical to those of p63. CONCLUSION: The MMp40 (BC28) monoclonal antibody is a high-quality screening antibody for determining squamous cell carcinoma in lung cancers, skin cancers of squamous or basal cell origin, squamous cell head and neck cancers, and urothelial carcinomas.

A new rabbit monoclonal E-cadherin antibody [EP700Y] shows higher sensitivity than mouse monoclonal E-cadherin [HECD-1] antibody in breast ductal carcinomas and does not stain breast lobular carcinomas.

Immunohistochemical studies have shown E-cadherin to be expressed in breast carcinomas showing a ductal histology, with a corresponding loss of expression in tumors with a lobular histology. As a result, mouse monoclonal anti-E-cadherin [HECD-1] has been used by pathologists to differentiate between ductal and lobular carcinomas, with currently published sensitivity and specificity rates of approximately 90%. Rabbit monoclonal antibodies may combine the best properties of both mouse monoclonal antibodies and rabbit antisera. Therefore, this study compares the staining sensitivity and specificity of a new rabbit monoclonal E-cadherin and the standard mouse monoclonal E-cadherin [HECD-1] in breast ductal carcinomas, and evaluates a cocktail of rabbit monoclonal E-cadherin and p120 catenin in the discrimination of ductal from lobular carcinomas. The rabbit E-cadherin showed sharper staining and increased sensitivity (80/81, 99%) than the mouse E-cadherin (75/81, 93%). The rabbit E-cadherin achieved a score of 3+ in 85.2% (69/81) of cases as compared with a 3+ in only 21.0% (17/81) of cases stained with mouse E-cadherin. All lobular carcinomas (n=37) were confirmed by the absence of E-cadherin and the diffuse cytoplasmic expression of p120 catenin. Although both the single mouse E-cadherin and dual stain can differentiate ductal from lobular lesions, the dual stain is helpful in challenging cases because of its bright pink p120 catenin and dark brown rabbit E-cadherin staining. The highly sensitive rabbit E-cadherin antibody is the preferred antibody for evaluating ductal carcinomas and for distinguishing ductal versus lobular lesions, and the dual stain was superior to the single E-cadherin stain.

Folate receptor α associated with triple-negative breast cancer and poor prognosis.

CONTEXT: Folate receptor α (FRA) has been shown to be selectively expressed in several types of human cancer, including breast cancer. Currently, several FRA target therapies are under intensive study. OBJECTIVE: To investigate the expression pattern of FRA in a large cohort of patients with breast cancer and analyze its relationship with different clinicopathologic features, with expression of several key biomarkers, and with clinical outcome. DESIGN: Four hundred forty-seven cases of infiltrating ductal carcinoma diagnosed between 1997 and 2008 at the University of Rochester Medical Center were identified and reviewed, and 25 blocks of tissue microassays were constructed. The association between expression of FRA and clinicopathologic features; expression of estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67; and clinical outcome of these tumors were evaluated. RESULTS: The expression of FRA was significantly associated with tumors with high histologic grade, higher nodal stages, ER/PR negativity, and high proliferative activity (Ki-67 ≥ 15%), and was independent of HER2/neu overexpression. In all, 74% of ER/PR-negative and 80% of triple-negative breast cancers expressed FRA. The expression of FRA was significantly associated with a worse disease-free survival. CONCLUSIONS: Our data demonstrate that a significant subgroup of ER/PR-negative and triple-negative breast cancers express FRA, and its expression is associated with worse clinical outcome.

Interobserver agreement and assay reproducibility of folate receptor α expression in lung adenocarcinoma: a prognostic marker and potential therapeutic target.

CONTEXT: Lung cancer is the leading cause of cancer deaths in the United States and globally. Folate-targeted drugs are among the promising new targeted therapies for lung cancer, provided predictive biomarkers can be identified for optimal patient selection. OBJECTIVE: To evaluate the interobserver agreement and reproducibility of an immunohistochemistry assay for folate receptor α as a potential predictive marker for folate-targeted therapies. DESIGN: Immunohistochemistry using anti-folate receptor α antibody 26B3 was performed on formalin-fixed, paraffin-embedded tissues. The M-score, a semiquantitative measure of staining intensity and proportion of tumor cells staining, was determined for each specimen. Interobserver agreement was assessed using lung adenocarcinoma specimens stained at a single site and evaluated by 3 independent pathologists. Interinstrument reproducibility assessed 20 specimens stained by 3 different automated stainers. Interlaboratory agreement was determined on 5 specimens, repeatedly stained on each of 5 days, at 3 different study sites. RESULTS: Folate receptor α expression was identified in 39 of 54 cases of lung adenocarcinoma (72%) and 4 of 37 cases of lung squamous cell carcinoma (11%). Agreement among 3 pathologists was found in 24 of 26 cases (92%). Interinstrument reproducibility was observed in 19 of 20 cases (95%). Agreement among 3 laboratories was found for 49 of 50 specimens (98%). CONCLUSIONS: Immunostaining of folate receptor α in lung adenocarcinomas is reproducible across staining platforms and among laboratories. Agreement among pathologists is achieved using a semiquantitative scoring method. An accurate and convenient method for determining folate receptor α expression offers a potentially invaluable tool for selecting patients for folate-targeted therapies.

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor.

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

PAX8 mouse monoclonal antibody [BC12] recognizes a restricted epitope and is highly sensitive in renal cell and ovarian cancers but does not cross-react with b cells and tumors of pancreatic origin.

PAX8 is expressed in a high percentage of renal cell and ovarian cancers; however, the current existing anti-PAX8 rabbit polyclonal (P) antibodies also recognize B cells, pancreatic cancers, carcinoids, and some soft tissue tumors. Cross-reactivity with B cells can be especially troublesome in lymph nodes when identifying tumors of unknown origin. A new mouse monoclonal (M) anti-PAX8 antibody (Clone BC12) has been developed that recognizes PAX8 expression in a high percentage of renal cell and ovarian carcinomas, whereas exhibiting no staining of B cells. PAX8 (M) was tested for specificity and sensitivity in over 1300 cases of both normal and neoplastic tissues. PAX8 (M) demonstrated superior staining sensitivity in clear cell and papillary renal cell carcinomas (88.8% vs. 84.4%) and in serous and endometrioid ovarian carcinomas (87% vs. 83%), when compared with PAX8 (P). PAX8 (M) also stained a high percentage of endometrial and thyroid cancers, 67.5% and 60.7%, respectively. PAX8 (M) demonstrated low sensitivity in cervical and bladder cancers, 2.5% and 1.4%, respectively. All other cancers including lung, breast, prostate, stomach, liver, soft tissue, pancreas, testis, brain, colon, melanoma, lymphoma, adrenal, pituitary, and rectal were negative. In normal tissue, PAX8 (P) stained lymph nodes, pancreas, and neuroendocrine cells of stomach and colon. In contrast, PAX8 (M) was negative in each of these tissues. These results demonstrate that mouse monoclonal PAX8 [BC12] stains nuclei exclusively and performs well in formalin-fixed paraffin-embedded tissues. PAX8 (M) is a highly sensitive marker for thyroid, renal, and ovarian cancers. Importantly, PAX8 (M) does not stain B cells and does not seem to recognize epitopes of pancreatic origin and neuroendocrine cells in stomach and colon; thus, providing superior specificity and making PAX8 [BC12] an excellent marker for confirming primary tumor site and for differential diagnosis.

A 6-antibody panel for the classification of lung adenocarcinoma versus squamous cell carcinoma.

Non-small cell lung cancer can be classified into several histologic subtypes, most commonly lung adenocarcinoma (LADC) or squamous cell carcinoma (SqCC). With the introduction of targeted therapies that can result in dramatically different outcomes based on subtype, the importance of accurate classification has been amplified. Six antibodies (Napsin A, Desmoglein 3, TTF-1, CK5, p63, and tripartite motif-containing 29) were selected for evaluation on cases of LADC of lung SqCC. Guided by the sensitivities and specificities determined for individual antibodies, a protocol was developed using a sequential series of 2-antibody cocktails that resulted in the classification of 93% of cases with 100% specificity. Importantly, the initial step in this method, a napsin A+Desmoglein 3 antibody cocktail classified >85% of cases, resulting in <15% of cases requiring further evaluation beyond a single test. Two new antibodies specifically developed and optimized for the diagnosis of LADC and lung SqCC, a rabbit polyclonal Napsin A and a mouse monoclonal Desmoglein 3 [BC11], were the key elements of the antibody panel. Most importantly, the described protocol uses routine interpretation methods and an uncomplicated algorithm for classification. Given the increased difficulty of diagnosing poorly differentiated tumors, the ability of this 6-antibody panel to classify 96% and 87% of moderately and poorly differentiated cases, respectively, is of particular value, especially when limited tissue for molecular testing is an issue.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.

Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase.

The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable, small-molecule inhibitor of the pim-1 kinase to foster basic and translational studies of the enzyme. We used an ELISA-based kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3,3',4',5,6,7-hydroxyflavone) was identified as a moderately potent, ATP-competitive inhibitor (IC(50), 0.34 micromol/L). Resolution of the crystal structure of PIM1 in complex with quercetagetin or two other flavonoids revealed a spectrum of binding poses and hydrogen-bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared with PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED(50), 5.5 micromol/L). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked PIM1 kinase activity. Furthermore, the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.

Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma.

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin's lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.